Exploiting Nanotechnologies and TRPV1 Channels to Investigate the Putative Anandamide Membrane Transporter
Figure 6
Effect of specific AEA cellular uptake inhibitors on PCL-NP-AEA mediated cellular uptake and [Ca2+]i elevation.
(A) PCL-NP-AEA (▪) exhibited similar potency and efficacy as “free” AEA (□) on [Ca2+]i in intact HEK-293 cells over-expressing the human recombinant TRPV1 receptor. The effect is expressed as percent of the effect on Ca2+ by ionomycin, 4 µM, and, in both cases, was dose-dependent and TRPV1-mediated because it was abolished by I-RTX (• and ○). (B) PCL-NP-AEA-induced elevation of [Ca2+]i in intact TRPV1-HEK-293 cells over-expressing the human TRPV1 receptor was significantly less sensitive to pharmacological tools previously used to specifically interfere with AEA cellular uptake or intracellular trafficking (BSA, OMDM1, MβCD, OA, FABP4 inhib). *p<0.05; ***p<0.001 compared to AEA-PCL-NP + vehicle (n = 3–5); #, ##, ### p<0.05, p<0.01 and p<0.005 compared with AEA+corresponding inhibitor (see Fig. 1A). (C) PCL-NP-AEA effect (full bars) on TRPV1-mediated [Ca2+]i was significantly less sensitive to concentrations of the selective AEA uptake inhibitors, OMDM-1 and AM1172, which instead strongly affected the effect of “free” AEA (dotted bars). *p<0.05; ***p<0.001 compared to the corresponding effect of the inhibitor observed using PCL-NP-AEA; # p<0.05 compared with PCL-NP-AEA + vehicle. In (B) data are expressed as percent of the maximal response observed with PCL-NP-AEA (1 µM) + vehicle (see panel (A)). In (C) data are expressed as percent of the maximal response observed with either PCL-NP-AEA (1 µM) + vehicle or AEA (1 µM) + vehicle (see panel (A)). Each bar indicates means ± sem of at least 3 independent experiments.